The functioning of neural circuits in the human brain is fundamentally important for understanding brain disease, psychiatric disorders, movement disorders, and addiction. Although mapping biostructures and the connectivity between neurons is important for understanding circuit structure, directly observing electrical communication within and amongst neurons is critical for complete understanding of neural circuitry function. Towards this end, second harmonic generation (SHG) microscopy has been developed for imaging neural activity because it can image relatively deep within biological tissue and because the reporting chromophores are directly responsive to membrane potentials. To date, the chromophores used in SHG imaging are asymmetric and are believed to operate via a Stark shift. Although these chromophores have provided very useful information about SHG imaging, their sensitivity appears to be limited by the large fields required for moderate Stark shifts, whih are about 10 to 100 times greater than a neuron's membrane potential. This limited sensitivity requires higher optical powers that can cause cell damage and even death. Herein we propose to take a somewhat counterintuitive approach by using chromophores that are symmetrical, and therefore have no SHG at the resting potential, but are converted to asymmetrical chromophores with large SHG signals and sensitivity in situ by the action potential. What is experimentally known about such symmetry breaking suggests that it may be possible to synthetically tune chromophores to the edge of symmetry breaking so that the process can be induced by an external stimulus such as the change in membrane potential. The program will combine quantum chemical modeling, computationally guided synthesis of chromophores that are compatible with biological media, and characterization of the chromophores in biologically relevant model membranes to more fully delineate and understand the molecular symmetry breaking and the SHG response. As chromophores are synthesized and tested, their response will be used to further refine the quantum models to more accurately guide subsequent synthesis. Chromophores identified as good candidates for SHG imaging will be studied in live neurons in the laboratories of Rafael Yuste at Columbia University. Although voltage-dependent symmetry breaking is largely untested experimentally, it has a strong theoretical basis, and so while there is significant risk, there is also potentially transformational reward associated with t. The ultimate goal of the program is to provide a new class of chromophores with much larger sensitivity so that a broad base of neuroscience researchers can practically and routinely use SHG imaging to map the functioning of neural circuits.